Spectrometry is the art of inferring information about an analyte based on its interaction with electromagnetic fields and radiation. Mass spectrometry, as its name suggests, is concerned with measurements of mass. Mass spectrometers (MS) have been called the smallest scales in the world because some of them can ‘weigh’ a single atom. Over time, the use of mass spectrometry has been expanded to larger and larger molecules, including macromolecules.
Nobel Laureate John B. Fenn remarked, “mass or weight information is sometimes sufficient, frequently necessary, and always useful in determining the identity of a species.” As mass spectrometry has become compatible with larger and larger analytes, this statement has remained true, with MS being used frequently to identify macromolecular components in biochemical mixtures. In the post-genomic era, there is more interest than ever in the characterization of increasingly massive macromolecular assemblies, and even larger bioparticles such as viruses and whole cells.
The masses of intact bioparticles, including viruses, bacteria, and whole mammalian cells have indeed been measured with mass spectrometers employing soft desorption techniques, such as laser-induced acoustic desorption (LIAD). A mass analyzer employing a trap may be used for light-scattering measurements to determine the mass-to-charge ratio (m/z) for these desorbed bioparticles. To determine masses of these bioparticles, the number of charges of the desorbed microparticles needs to be changed by electron bombardment in order to observe changes in their light-scattering patterns. One problem with this approach is that this process of changing the number of charges can be excessively time consuming. For example, on average it takes about 15-30 minutes to determine the mass of one trapped microparticle. As mass distributions of most bioparticles are broad and many microparticles have to be measured to obtain a mass distribution, it becomes impractical to perform conventional light-scattering techniques for mass-distribution measurements of microparticles.
An additional problem with previous approaches relates to noise levels and precision of charge measurement. A single microparticle can have charge numbers in the range of 10-2,000 under matrix-assisted laser desorption-ionization (MALDI) or LIAD measurement processes. However, accurate determination of mass by direct measurement of the number of charges on these desorbed cells or microparticles has been a challenge because of the low number of charges on the cells or microparticles relative to electronic noise due to the detection apparatus.
In most conventional mass spectrometers, ions are detected by a charge-amplification device, such as a microchannel plate (“MCP”). Because the charge-amplification device detects charges based on ejection of secondary electrons, this type of detector is typically associated with an undesired detection bias. Moreover, the efficiency of secondary-electron ejection is closely related to the velocity of the incoming ions. Therefore, mass spectra of mixtures of large biomolecules usually do not reflect the actual number of ions detected at a charge-amplification device.